The opioid peptide neurotransmitter dynorphin is synthesized through the proteolysis of the larger prodynorphin precursor protein. Dynorphin has a well-documented role in modulating pain and mechanisms involved in substance abuse. A better understanding of these important processes could therefore emerge from elucidation of the proteolytic pathways through which inactive prodynorphin is cleaved to produce active dynorphin peptides. Two primary protease pathways are implicated in the synthesis of mature peptide neurotransmitters, or neuropeptides, consisting of the recently characterized cathepsin L cysteine protease in secretory vesicles, together with the well-known proprotein convertase (PC) proteases PC 1/3 and PC2. New results demonstrate participation of the novel cathepsin L cysteine protease pathway in secretory vesicles for dynorphin biosynthesis. Notably, cathepsin L gene knockout mice show that dynorphin levels in brain are reduced by 75%. Furthermore, expression of cathepsin L results in dynorphin production in PC12 cells. Based on these new findings, it is hypothesized that cathepsin L is an important processing protease for dynorphin production. The goal of this proposal is to evaluate the production of dynorphin from prodynorphin by cathepsin L This goal will be achieved in three specific aims. Specific aim 1 will assess dynorphin peptide production by cathepsin L during coexpression of prodynorphin and cathepsin L in neuroendocrine PC12 cells, combined with cellular evaluation of cathepsin L colocalization with dynorphin in secretory vesicles of the regulated secretory pathway. Complementary in vivo studies of cathepsin L will be achieved in specific aim 2 which will assess dynorphin and prodynorphin-derived products in cathepsin L gene knockout mice, with comparisons to PC1/3 and PC2 knockout mice. Results will indicate the impact of each of these processing proteases on dynorphin neuropeptide production. Specific aim 3 will assess the effects of a selective inhibitor of cathepsin L during dynorphin production in chromaffin cells and brain cortical neurons in primary culture. Results will indicate how cellular systems utilize the cathepsin L processing pathway for production of dynorphin. Dynorphin plays an important role in modulating pain, including chronic pain, and neuroplasticity associated with drug abuse. Knowledge gained from this study will increase our understanding of these processes and provide targets for regulatory agents that may modulate dynorphin production to facilitate its beneficial effects or reduce its adversive effects.